Molecular basis for PrimPol recruitment to replication forks by RPA.

DNA damage and secondary structures can stall the replication machinery. Cells possess numerous tolerance mechanisms to complete genome duplication in the presence of such impediments. In addition to translesion synthesis (TLS) polymerases, most eukaryotic cells contain a multifunctional replicative enzyme called primase-polymerase (PrimPol) that is capable of directly bypassing DNA damage by TLS, as well as repriming replication downstream of impediments. Here, we report that PrimPol is recruited to reprime through its interaction with RPA. Using biophysical and crystallographic approaches, we identify that PrimPol possesses two RPA-binding motifs and ascertained the key residues required for these interactions. We demonstrate that one of these motifs is critical for PrimPol's recruitment to stalled replication forks in vivo. In addition, biochemical analysis reveals that RPA serves to stimulate the primase activity of PrimPol. Together, these findings provide significant molecular insights into PrimPol's mode of recruitment to stalled forks to facilitate repriming and restart.

Primase-polymerases are a functionally diverse superfamily of replication and repair enzymes.

Until relatively recently, DNA primases were viewed simply as a class of proteins that synthesize short RNA primers requisite for the initiation of DNA replication. However, recent studies have shown that this perception of the limited activities associated with these diverse enzymes can no longer be justified. Numerous examples can now be cited demonstrating how the term 'DNA primase' only describes a very narrow subset of these nucleotidyltransferases, with the vast majority fulfilling multifunctional roles from DNA replication to damage tolerance and repair. This article focuses on the archaeo-eukaryotic primase (AEP) superfamily, drawing on recently characterized examples from all domains of life to highlight the functionally diverse pathways in which these enzymes are employed. The broad origins, functionalities and enzymatic capabilities of AEPs emphasizes their previous functional misannotation and supports the necessity for a reclassification of these enzymes under a category called primase-polymerases within the wider functional grouping of polymerases. Importantly, the repositioning of AEPs in this way better recognizes their broader roles in DNA metabolism and encourages the discovery of additional functions for these enzymes, aside from those highlighted here.

Human PrimPol mutation associated with high myopia has a DNA replication defect.

PrimPol is a primase-polymerase found in humans, and other eukaryotes, involved in bypassing lesions encountered during DNA replication. PrimPol employs both translesion synthesis and repriming mechanisms to facilitate lesion bypass by the replisome. PrimPol has been reported to be a potential susceptibility gene associated with the development of myopia. Mutation of tyrosine 89 to aspartic acid (PrimPolY89D) has been identified in a number of cases of high myopia, implicating it in the aetiology of this disorder. Here, we examined whether this mutation resulted in any changes in the molecular and cellular activities associated with human PrimPol. We show that PrimPolY89D has a striking decrease in primase and polymerase activities. The hydrophobic ring of tyrosine is important for retaining wild-type extension activity. We also demonstrate that the decreased activity of PrimPolY89D is associated with reduced affinities for DNA and nucleotides, resulting in diminished catalytic efficiency. Although the structure and stability of PrimPolY89D is altered, its fidelity remains unchanged. This mutation also reduces cell viability after DNA damage and significantly slows replication fork rates in vivo. Together, these findings establish that the major DNA replication defect associated with this PrimPol mutant is likely to contribute to the onset of high myopia.

Molecular dissection of the domain architecture and catalytic activities of human PrimPol.

PrimPol is a primase-polymerase involved in nuclear and mitochondrial DNA replication in eukaryotic cells. Although PrimPol is predicted to possess an archaeo-eukaryotic primase and a UL52-like zinc finger domain, the role of these domains has not been established. Here, we report that the proposed zinc finger domain of human PrimPol binds zinc ions and is essential for maintaining primase activity. Although apparently dispensable for its polymerase activity, the zinc finger also regulates the processivity and fidelity of PrimPol's extension activities. When the zinc finger is disrupted, PrimPol becomes more promutagenic, has an altered translesion synthesis spectrum and is capable of faithfully bypassing cyclobutane pyrimidine dimer photolesions. PrimPol's polymerase domain binds to both single- and double-stranded DNA, whilst the zinc finger domain binds only to single-stranded DNA. We additionally report that although PrimPol's primase activity is required to restore wild-type replication fork rates in irradiated PrimPol-/- cells, polymerase activity is sufficient to maintain regular replisome progression in unperturbed cells. Together, these findings provide the first analysis of the molecular architecture of PrimPol, describing the activities associated with, and interplay between, its functional domains and defining the requirement for its primase and polymerase activities during nuclear DNA replication.